Anatomically, the mammalian adrenal cortex is composed of three concentric zones. The aim of this project is to study the development of adrenocortical zonation and to examine, in depth, differential function and regulation of the separate zones. Various approaches have been employed using whole tissue, isolated cells, and tissue culture. The guinea pig, a cortisol producer like the human being, is being utilized as an animal model because of the relative ease in grossly separating the innermost zone (reticularis) from the outer two zones (glomerulosa and fasciculata). Cells from the latter two zones can be further separated by more refined techniques, eg., elutriation, sedimentation at unit gravity, or density gradient centrifugation. Specific cells, either acutely isolated or in tissue culture, are being examined to determine their steroidogenic and nucleotide responses to various hormonal and environmental manipulations. Isolated plasma membranes are being studied in regard to peptide hormone and lipoprotein receptor activity, regulation of the adenylate cyclase system, phospholipid turnover, and ATPase activity. The rate-limiting step in steroidogenesis is mitochondrial conversion of cholesterol to pregnenolone. Extensive experimentation is in progress to examine this key enzyme system using purified mitochondria from different zones. The latter studies are being coordinated with measurement of mitochondrial cholesterol. In addition to the cholesterol lyase reaction, all the steroid biosynthetic enzymes and certain metabolic enzymes, such as the 3Beta-steroid sulfotransferase, are being investigated. The volume and relative proportion of the zona reticularis increases dramatically with age for reasons that are not at understood. This phenomenon occurs in both the human being and the guinea pig. Areas of research that should enhance our understanding of adrenocortical zonation and function have to do with developmental factors and differential regulation, and the guinea pig should continue to be a useful animal model for examining these important questions.